Acyl-CoA has been proposed as a regulator of heart citrate synthase in vivo. We have prepared spin-labeled acyl-CoA (doxylstearyl-CoAs) and are studying the electron spectra of these acyl-CoA analogues in solution (micelles) and associated with citrate synthase. The bound spectra show that an acyl spin label near the polar portion of the molecule is more strongly immobilized. Binding to citrate synthase can be observed below 1 mole acyl-CoA per mole enzyme up to more than 15 moles per mole. The binding is decreased in the following order: oxalacetic acid greater than NADPH greater than ATP greater than acetyl-CoA. Thus, the CoA binding sites are not shared. This data supports the suggestion that a specific binding site for acyl-CoA may exist. Acyl-CoA is very strongly partitioned into phospholipid; this partition can be directly quantitated by the difference in spectra between 6-doxylstearyl-CoA in bilayers and free in solution. Acyl-CoA in vivo is most likely a normal membrane component.